pharmaceuticals

Review
Semi-Solid and Solid Dosage Forms for the Delivery
of Phage Therapy to Epithelia
Teagan L. Brown 1 ID
, Steve Petrovski 2 , Hiu Tat Chan 3 , Michael J. Angove 1 and Joseph Tucci 1, *
1 Department of Pharmacy and Applied Science, La Trobe Institute for Molecular Science, La Trobe University
Bendigo Campus, PO Box 199, Bendigo 3550, Australia; teagan.brown@latrobe.edu.au (T.L.B.);
m.angove@latrobe.edu.au (M.J.A.)
2 Department of Physiology, Anatomy and Microbiology, La Trobe University Bundoora,
Bundoora 3083, Australia; steve.petrovski@latrobe.edu.au
3 Department of Microbiology, Royal Melbourne Hospital, Parkville 3050, Australia; mark.chan3@mh.org.au
* Correspondence: j.tucci@latrobe.edu.au; Tel.: +61-3-5444-7897

Received: 19 December 2017; Accepted: 23 February 2018; Published: 26 February 2018

Abstract: The delivery of phages to epithelial surfaces for therapeutic outcomes is a realistic proposal,
and indeed one which is being currently tested in clinical trials. This paper reviews some of the known
research on formulation of phages into semi-solid dosage forms such as creams, ointments and pastes,
as well as solid dosage forms such as troches (or lozenges and pastilles) and suppositories/pessaries,
for delivery to the epithelia. The efficacy and stability of these phage formulations is discussed,
with a focus on selection of optimal semi-solid bases for phage delivery. Issues such as the need for
standardisation of techniques for formulation as well as for assessment of efficacy are highlighted.
These are important when trying to compare results from a range of experiments and across different
delivery bases.

Keywords: phages; phage therapy; phage formulation

1. Introduction
An important clinical concern today is that of microbial resistance to commonly used
chemotherapeutic agents for infectious disease. Although resistance to antibiotics was documented in
the first half of the 20th century, their indiscriminate use and inappropriate application has seen the
frequency of resistance escalate [1]. As the number of antibiotic resistant bacteria rapidly grows, the
need to find new antimicrobial agents becomes crucial to prevent health care systems from reverting
back to the pre-antibiotic era [2]. Antibiotic resistance poses a significant threat to human health, as
well as increasing healthcare costs worldwide [2,3]. The issue is recognised as a worldwide crisis
needing urgent attention, and, while the instigation of Public Health infectious control and antibiotic
stewardship programs have been advantageous, these should be complemented with new approaches
and initiatives [1,2,4–6].
An alternative to antibiotics is the use of lytic phages to eliminate bacterial infection [7]. The first
reported use of phage therapy, prior to 1920, was by Felix d’Herelle, the co-discoverer of phage.
He applied phages in the treatment of plague and cholera [7,8]. In the early 1920s, the treatment of
patients with staphylococcal skin infections, where the phages were injected into and around surgically
opened lesions, was published [9,10]. Yet despite these efforts and interest surrounding phage therapy,
research in the area was abandoned in Western countries for the newly discovered and convenient
antibiotics in the 1940s [11]. Work in phage therapy continued in former Eastern bloc countries and
only recently has become more popular worldwide [12]. There are many benefits of using phage in
therapy. Their host specificity leads to a targeted lysis of the bacteria involved in the infection and even
some biofilms [13]. Further, they are auto “dosing” as their replication leads to an increased titre at the

Pharmaceuticals 2018, 11, 26; doi:10.3390/ph11010026 www.mdpi.com/journal/pharmaceuticals

Pharmaceuticals 2018, 11, 26 2 of 12

site of infection and they display single hit kinetics. Importantly, phages have low inherent toxicity,
and are generally regarded as safe by the US Food and Drug Administration [13]. Additionally, phages
will lyse antibiotic resistant strains and are less likely to bring about resistance [13,14]. From 2016, the
National Institutes of Health (USA) have funded phage therapy research projects, with the view that
these non-traditional therapies could provide strategies to combat anti-microbial resistance [15].
As with pharmaceutical drug delivery, targeting phages to the site of infection remains a hurdle
for efficient therapy [12]. There are specific issues that need to be considered when using phages for
clinical purposes. For instance, phages are comparatively large biological entities that require structural
integrity and viability for efficacy. This requirement poses additional challenges (as compared to
delivery of antibiotics/antiseptics which are small chemical molecules) during the formulation process
and storage, as well as in the design of protocols for efficacy testing. On the other hand, their ability to
replicate results in increased levels at the site of infection, compared to the delivered concentration.
This is in direct contrast to antibiotics administrated systemically, whose concentration at the site of
infection is significantly less than that administered. Further, while topical antibiotics are usually
not recommended because of the risk of resistance development [16], topical phage therapy can be
desirable. As such, infection which previously required systemic antibiotic therapy such as skin, soft
tissue and surgical site infection can be potentially treated with topical phage therapy. These novel
strategies can also be investigated in applications such as topical surgical prophylaxis.
To date, a diverse range of applications for phage therapy have been reported. Most commonly,
liquid preparations are utilised as dosage forms for injections (cutaneous, intravenous, subcutaneous,
intrapleural) and local application [14,17]. A wide range of in vitro and animal models have shown
that delivery of phages for control of bacterial growth is effective in a range of experimental
systems. In mouse models, phages have been delivered by diverse routes such as intraperitoneal
injection of a three-phage cocktail for treatment of Klebsiella pneumonia [18], intraperitoneal injection
of Podoviridae phages for Cronobacter turicensis urinary tract infection [19], and by subcutaneous
injection of Podoviridae phages in treatment of Escherichia coli pneumonia, sepsis and urinary tract
infections [20]. In rainbow trout and zebrafish, Columnaris disease was treated with Myoviridae phages
lytic for Flavobacterium columnare in the aqueous habitat [21]. In rabbit models of osteomyelitis caused
by Staphylococccus, the intraperitoneal injection of a seven-phage cocktail showed efficacy [22] and
the intranasal administration of Podoviridae viruses lytic for Pseudomonas aeruginosa was effective
in ameliorating haemorrhagic pneumonia in Mink [23]. Yet more basic research as well as the
accumulation of data from extensive clinical trials is required in order to provide comprehensive
evidence which will allow approval of phage therapy by regulatory bodies such as the US Food
and Drug Administration and the European Medicines Agency [24,25]. This paper reviews some of
the known data regarding the formulation of phages in semi-solid and solid dosage forms for the
targeting of bacteria associated with epithelial surfaces. It discusses specific issues relating to these
formulations and highlights the need for some standardisation in assessment of efficacy of these
delivery mechanisms.

2. Formulations of Semi-Solid and Solid Dosage Forms for Delivery to Epithelia

Semi-solid emulsions such as creams and ointments are very useful in the delivery of medicaments
to epithelial surfaces such as the skin. They tend to be minimally irritating on the skin, easy to apply,
easily removed with soap and water, stable enough to avoid the need for frequent applications,
and bacteriostatic [26]. Pastes are semi-solid carriers which are often “fatty” and quite stiff in
consistency [27]. Their use is often in the treatment of oozing lesions, where their heavy consistency
confers a degree of physical protection, and absorptive properties allow them to absorb secretions
from wounds. From a patients’ perspective, the more hydrophilic bases may be preferred as vehicles
for therapy applied to the facial region, as they are easier to apply and less greasy than the more
hydrophobic ointments. The thicker pastes are indicated where there is the need for application to
Pharmaceuticals 2018, 11, 26 3 of 12

moist surfaces such as oozing wounds, or inside the oral cavity, where other vehicles would be easily
removed [27].
Suppositories are solid dosage forms, tapered at one end, which can carry medicaments into
epithelial cavities such as the rectum, vagina or urethra. After insertion, these formulations become
soft and disperse. Troches (also known as lozenges or pastilles) are solid dosage forms for the delivery
of medication orally. They can deliver medicaments to epithelial surfaces such as the oral cavity,
oesophagus and gut. They are placed in the mouth, and at temperatures approaching 37 ◦ C they
slowly dissolve, liberating the active ingredient [27].
As with the formulation of any pharmaceutical drug into a semi-solid dosage form, it is important
that phages are incorporated into the vehicle such that there is homogenous distribution throughout
the final product. This allows mixture uniformity and ensures consistent delivery of the medicament.
In industry this process is undertaken using large scale mixing equipment [27,28], but for the
preparation of phage formulations in semi-solid and solid dosage forms as described here for research
purposes, the reliance is on small scale mixing equipment and strategies. If results of research into
delivery of phage therapy by semi-solid and solid dosage forms are to be reliable, then the outcomes of
the small scale mixing processes should be consistent with those of large scale mixing, that is, mixture
uniformity, to ensure consistent delivery of the phages. An important issue, however, is that phage
structures (e.g., phage tails) may be compromised during large scale mixing processes. Therefore,
large scale manufacturing processes have to be verified by industry to demonstrate phage efficacy
equivalent to small scale "in-house" mixing processes in research settings.
Geometric dilution is a commonly used technique when low-dose active pharmaceutical
ingredients (API) are blended into formulations. It implies the gradual addition of equal portions of the
diluent to the API upon blending [29]. The process is an effective way to enhance the equal distribution
of the API within the blend, and an increase in mixing time promotes better distribution of the active
ingredient [30]. In the formulation of small volumes of concentrated phage solution into semi-solid
vehicles, as reported by Brown et al. [31], the process involves the addition, mixing (for at least five
minutes) and even distribution of the phage solution into a small portion (approximately 1–2 g) of the
semi-solid vehicle, then thorough mixing (for at least five minutes) of this small portion with an equal
mass of fresh vehicle. This process is repeated until all the fresh cream has been incorporated, and
ensures that the medicament (in this case, the phage) is evenly dispersed throughout the cream. The
mixing of phages into solid dosage forms such as troches and suppositories present specific issues,
which will be discussed below.

3. Stability of Phages in Semi-Solid Preparations

In the west reports of testing of a phage in a semi-solid preparation suggested that Myoviridae
phages lytic for Staphylococcus aureus incorporated into a commercial cream (at a concentration of
108 PFU per gram) were capable of clearing bacterial cells in broth following insertion of a strip of the
cream and a four-hour incubation period at 37 ◦ C [32]. Adequate controls for these experiments were
not shown, and it was unclear whether preservatives within the bismuth-based commercial cream
had any effect on bacterial growth. In a subsequent study, a commercial cream containing paraffin
mineral oil was diluted with water to obtain a lotion, into which were mixed Podoviridae phages lytic
for Acinetobacter baumannii (at a concentration of 108 PFU per gram). In these experiments there was
no discussion on the possible effects of preservatives in the commercial cream on bacterial growth.
The results showed that while such a lotion was capable of killing bacteria, the lytic capacity was not
maintained after 30 days storage of the phage lotion at room temperature [33]. More recent studies had
combined commercial burn wound care products with Myoviridae and Podoviridae phages which were
lytic for A. baumannii, P. aeruginosa and S. aureus, then assessed the effect on phage stability and viability
following 24 h at 37 ◦ C. These commercial products included creams, gels, suspensions and ointments,
and assays involved the dilution of these products 1:1 with phage suspensions. Therefore, the study
did not investigate the capacity of intact semi-solid preparations to deliver viable phages, but did
Pharmaceuticals 2018, 11, 26 4 of 12

demonstrate that some of the active ingredients in the commercial products, such as antibacterial agents,
and in particular, acidic compounds, had an adverse effect on phage viability [34]. While this study
provided important insight into possible issues when formulating phages into commercial preparations
for bacterial control, it highlighted the potential difficulty in dissociating phage effects from other
antibacterial effects when such formulations are used. Also, the issue of overuse of antiseptics and
antibiotics and potential spread of antimicrobial resistance in bacteria is not addressed with such
formulations. To this end, the current phagoburn Phase I/II clinical trials (NCT02116010) which aim
to treat burns victims with wound infections caused by E. coli and P. aeruginosa are employing phage
cocktails as the sole antimicrobial agents [35,36].
It is important to ascertain the stability of phage preparations in semi-solid formulations in order
to deliver efficacious phage therapy in such carriers. Testing of these formulations should incorporate
assays to determine thermo-stability and photo degradation over time [27–29]. The most extensive
stability tests of such phage formulations that have been reported to date have included storage of
the formulations at 4 ◦ C, 20–25 ◦ C and 45 ◦ C in light-protected bottles to ascertain the thermostability
and exposure to 50 Lux of light, the standard illumination of a typical room, at 20–25 ◦ C to test the
phages photodegradation over time [37]. The quantitation of phage lytic activity following such
assays is important. A relatively straightforward quantitative method for this is to measure a standard
small weight of the phage cream preparation, and then dilute this into an aqueous solvent such that
there is even dispersal of the phage particles throughout. The numbers of active phage particles
can then be determined by serial dilution and counting of plaques on a bacterial lawn [37]. One
of the issues, however, is that such a method relies on the miscibility of all the components of the
semi-solid preparations in the solvent, and so may be more suitable for phage formulations in more
hydrophilic creams [31]. The use of a benign aqueous solvent (e.g., water, physiological saline or
phosphate buffered saline (PBS)) is important here, as other solvents may prove toxic to bacteria
growing on agar plates or the phages contained in the formulation. In more lipophilic ointments, such
even dispersal of the components in the aqueous solvent is difficult, and so any count of phage plaques
may be skewed, and therefore not a true representation of particle numbers (see below). An alternative
method for quantitation may involve measurement of zones of clearing surrounding phage cream
strips placed upon a bacterial lawn, as employed by agar diffusion tests in the determination of
antimicrobial efficacy.
Agar diffusion tests such as the Kirby-Bauer disc diffusion method are well established and
adopted by International bodies such as the Clinical Laboratory Standards Institute (CLSI) and the
European Committee on Antimicrobial Susceptibility Testing (EUCAST) [38]. Such assays require
strict standardisation of parameters including diffusion of antimicrobial agent, interaction between
media and antimicrobial agent, hydration of media etc. and bodies such as CLSI are instrumental in
establishing guidelines to guarantee quality control in such methods [39,40]. The rigorous scientific
assessment of phage stability and efficacy in semi-solid formulations is as yet in its infancy, and not
subject to such stringent guidelines. Yet standardisation of phage stability and activity in semi-solid
preparations is an important issue, and one which requires more research effort to ensure the efficacy
of future phage therapy applications using such carriers. It is important to note, however, that the
antibiotic testing methodologies mentioned above are for testing purified antibiotic molecules only, and
not antibiotics in formulations. Regulatory bodies may adopt current guidance such as potency testing
for cellular therapy products and biologics to guide development of standards for bacteriophage
formulation efficacy testing [41].
The quantitative assessment of phage stability and lytic activity over time (determined by counting
of plaques following mixing of small amounts of phage cream 1:100 into PBS, as described above),
were employed for Propionibacterium acnes Siphoviridae phages formulated into more hydrophilic
cream bases (cetomacrogol cream aqueous, aqueous cream, and cetrimide cream aqueous) [31]. These
assessments showed that storing the formulations at 4 ◦ C in light-protected containers resulted in
the greatest levels of viable P. acnes phages after 90 days, and that the next best conditions were
Pharmaceuticals 2018, 11, 26 5 of 12

storage at room temperature (20–25 ◦ C) in a light-protected container [37,42]. Storage at 45 ◦ C and at

constant light at 25 ◦ C resulted in a rapid decline in efficacy for the P. acnes phage creams (Figure
Pharmaceuticals 2018, 11, x
1).
5 of 12
Cetomacrogol cream aqueous provided optimal retrieval of lytic P. acnes phage numbers in these
quantitation
these stability assays
tests(Figure 2 and Table 1).
assess Siphoviridae It is important
phages specific fortoP.
note, however,
acnes, and other thatphages
these stability
may behavetests
assess Siphoviridae phages specific for P. acnes, and other phages may behave
differently in these formulations. These quantitation assays also do not necessarily reflect the capacitydifferently in these
formulations.
for the cream These quantitation
to adequately deliverassays alsofor
phages dotherapy.
not necessarily reflectafter
For instance, the capacity for the cetrimide
90-day storage, cream to
adequately deliver phages for therapy. For instance, after 90-day storage,
cream scored more highly than aqueous cream in quantitation assays, yet after this time, although cetrimide cream scored
more highlycream
cetrimide than aqueous cream in quantitation
was a repository of viable P. assays, yet after
acnes phage this time,
particles, although
these cetrimide
were shown to cream
not be
was a repository
released viable P. acnes
and lyseofunderlying phage particles,
and surrounding thesewhen
bacteria weretheshown
cream towas
not placed
be released
onto aand lyse
P. acnes
underlying
bacterial lawn on agar (Brown, unpublished data; see discussion in Section 4 for elaboration).on
and surrounding bacteria when the cream was placed onto a P. acnes bacterial lawn In
agar (Brown, P.
comparison, unpublished
acnes phages data; see discussioncream
in cetomacrogol in Section
aqueous4 forwere
elaboration).
released and In comparison,
lysed bacterialP. acnes
cells
phages
when ain cetomacrogol
strip of the phage cream
cream aqueous wereupon
was placed released
a P. and
acneslysed bacterial
bacterial lawncellson anwhenagaraplate
strip(Figure
of the
phage cream was placed upon a P. acnes bacterial lawn on an agar plate (Figure
3), and this clearing was seen even following 90-day storage at 4 °C. These authors also showed that 3), and this clearing
was
when seen even phages
P. acnes following 90-day
were storage
triturated into 4 ◦ C. These authors
at formulations also showed
of cetomacrogol when P. that
thataqueous
cream acneshadphages
been
were
prepared with and without preservative (0.1% chlorocresol), there was minimal effect ofand
triturated into formulations of cetomacrogol cream aqueous that had been prepared with the
without preservative
preservative (0.1%survival,
on bacterial chlorocresol),
and on there
phagewaslytic
minimal effect
activity and of efficacy
the preservative on bacterial
(Brown, unpublished
survival,
data). and on phage lytic activity and efficacy (Brown, unpublished data).

Figure1.1.Quantitative
Figure Quantitativeassessment
assessmentof ofP.
P.acnes
acnesphage
phage(PAC1)
(PAC1)lytic
lyticactivity
activityand
andstability
stabilityin
incetomacrogol
cetomacrogol
cream aqueous following storage at various temperatures and light exposures
cream aqueous following storage at various temperatures and light exposures [31,43]. Log scale [31,43]. Log scale
shown,
shown, with data points representing the mean of three samples. ● indicates ◦
storage
with data points representing the mean of three samples. indicates storage at 4 C in a light protectedat 4 °C in a light
protected
bottle; bottle; ○
# indicates indicates
storage at 20–25 ◦
storage
C inata20–25 °C in a light
light protected bottle; ∆ indicates
protected bottle; Δ indicates
storage in constantstorage in
light at
20–25 ◦ C; light
constant and H atindicates and ▼ at
20–25 °C;storage 45 ◦ C instorage
indicates a light at 45 °C inbottle.
protected a light protected bottle.

Table1.1. P.
Table P. acnes
acnes phage
phage PAC1
PAC1formulated
formulatedinto
intosemi-solid
semi-solidcreams.
creams. Mean
Mean differences
differences in
in phage
phage
concentration (log ) in the various cream types after storage for 90 days at 4 ◦°C. For P. acnes
concentration (log10 ) in the various cream types after storage for 90 days at 4 C. For P. acnes phage
10 phage
PAC1cetomacrogol
PAC1 cetomacrogolwas wasthe
theoptimal
optimalsemi-solid
semi-solidpreparation
preparationtested
tested[31].
[31].

Cream (I) Cream (J) Mean Difference (I–J)Standard

Standard Error p-Value
p-Value
Cream (I) Cream (J) Mean Difference (I–J) Error
Aqueous
Aqueous Cetomacrogol
Cetomacrogol −1.329
−1.329 0.0800.080 <0.001
<0.001
Aqueous
Aqueous Cetrimide
Cetrimide −0.868
−0.868 0.0800.080 <0.001
<0.001
Cetomacrogol Cetrimide 0.461 0.078 <0.001
Cetomacrogol Cetrimide 0.461 0.078 <0.001
Pharmaceuticals 2018, 11, x 6 of 12
Pharmaceuticals 2018, 11, 26 6 of 12
Pharmaceuticals 2018, 11, x 6 of 12

Figure 2. P. acnes phage PAC1 stability in semi-solid dosage forms. These regression plots show 4 °C
P. acnes phage PAC1 ◦C
Figure
Figure 2. P.
(as depicted
2. by the
acnes circles)
phage PAC1andstability
20–25 °C
stability in semi-solid
in(as depicteddosage
semi-solid by the forms.
dosage squares) These regression
treatments
forms. These plots
for each
regression show type.
plotscream
show 4
4 °C
(as depicted by thein
circles) and 20–25 ◦ C (as depicted by the squares) treatments for each cream type.
Thedepicted
(as central bylines
the each and
circles) plot20–25
are the
°C regression
(as depictedbest fit squares)
by the lines and the curved
treatments for lines definetype.
each cream 99%
The central
confidence lines
bounds.in each
For plot
P. are
acnesthe regression
phage PAC1 best fit lines
cetomacrogol andwasthe curved
the lines
optimal define 99%
semi-solid
The central lines in each plot are the regression best fit lines and the curved lines define 99% confidence
preparation
bounds.
tested [31].
confidence P. acnes For
Forbounds. phage PAC1phage
P. acnes cetomacrogol was the optimal
PAC1 cetomacrogol wassemi-solid
the optimal preparation
semi-solidtested [31].
preparation
tested [31].

Figure 3. Lytic
Figure 3. Lytic capacity
capacity of the P.
of the P. acnes
acnes phage
phage PAC1
PAC1 cream formulation. P.
cream formulation. P. acnes
acnes phage
phage formulated
formulated in in
cetomacrogol
cetomacrogol
Figure cream
3. Lyticcream aqueous
aqueous
capacity on
of theon a lawn
P. aacnes of
lawnphage P. acnes
of P. acnes bacteria
PAC1bacteria [37]: (A) cetomacrogol
[37]: (A) cetomacrogol
cream formulation. cream
cream
P. acnes phage aqueous;
aqueous; (B)
formulated in
(B) cetomacrogol cream aqueous ×3 10 3
cetomacrogol
cetomacrogol cream
cream aqueous
aqueous with
on awith
PBS;
lawn PBS;
of (C)
(C)P.
phagephage
acnes cream,
cream,
bacteria PAC1
PAC1
[37]: at aatcetomacrogol
(A) a concentration
concentration of 5.0
of 5.0
cream PFUPFU
×aqueous;
10 per
(B)
per gram of cream; × 8108 PFU per gram of cream.
gram of cream;
cetomacrogol andand
cream (D) (D)
aqueous phage
phage with cream,
cream,
PBS;PAC1PAC1
(C) at aatconcentration
phage acream,
concentration
PAC1 of atof
2.52.5
× 10 PFU
a concentration perofgram of 3cream.
5.0 × 10 PFU per
gram of cream; and (D) phage cream, PAC1 at a concentration of 2.5 × 108 PFU per gram of cream.
Pharmaceuticals 2018, 11, 26 7 of 12

4. Effect of the Ionic Nature of the Semi-Solid Base

Determination of which semi-solid formulation is most suitable for the incorporation of phages
is important. Early studies suggested that phages are negatively charged from pH 3.6 to 7.6 [44].
Agarose gel electrophoresis of T7 Podoviridae phages demonstrated that the capsids held a negative
charge whereas the tail fibres were positively charged according to their migration [45,46]. The
immobilisation of phages onto silica particles through electrostatic interactions has shown that
Myoviridae and Siphoviridae phages targeting Escherichia coli, Salmonella enterica, Listeria monocytogenes
and Shigella boydii adsorb to positively charged silica surfaces [47]. The authors hypothesised that the
negatively charged capsids bound to the silica surface, “head down” such that the tails were able to
bind the bacteria. The biocontrol of Listeria and Escherichia via immobilisation of phages onto cationic
cellulose membranes [48] also suggested the capacity for binding to cations by the negative capsid.
The results indicated that the phages capsids were attached to the membrane and the tail fibres were
available to target the bacteria. Based on these findings, we surmised that the Siphoviridae [31,37] and
Myoviridae (Brown, unpublished data) phages used in our experiments would contain charged capsids
and tails, and as such there may be interactions between the phages and cream formulations. Ionic
polymers within some cream bases have the potential to interact with phages through electrostatic
forces. Therefore, the ionic nature of the cream may be important in terms of allowing “release”
of the phages to access the bacteria in underlying tissue upon which it is spread. In the testing
of cetomacrogol cream aqueous (a non-ionic base), aqueous cream (an anionic base) and cetrimide
cream aqueous (a cationic base) as carriers of phages, quantitative analysis of viable P. acnes phage
numbers following storage for 90 days showed that viable phage numbers in cetomacrogol cream
were significantly higher than in either aqueous or cetrimide creams (p-values < 0.001). Under the
same conditions, viable P. acnes phage numbers in cetrimide cream were significantly higher than
in aqueous cream (p < 0.001) [31]. It is important to note, however, that such quantitative analysis
involves dispersion and 1:100 dilution of the phage cream in aqueous buffer, then assessment of viable
phage by plaque numbers. Dispersion of the cream matrix in an aqueous solution is likely to change
the release profile of the phage, as the interaction of dispersed polymeric ions from the cream and
phage will be weaker than the interactions of phage within the polymer network of a non-dispersed
cream. As such, quantitative analysis may reflect the “storage” of the phages in the cream matrix, and
not necessarily the capacity of phage to be released from the cream and to lyse underlying bacteria.
Evidence of this was seen following formulation of P. acnes phages into the cationic cetrimide cream,
where while there may be clearing of the bacterial lawn initially after formulation, this clearing was
not seen when a sample of the cream was placed upon the bacterial lawn a week or so later (Brown,
unpublished data), even though quantitative analysis showed that there were still viable P. acnes
phages present in the cream. Therefore, it would seem prudent to assess phage stability in creams by
quantitative measures of their viability following storage over time, as well as qualitative functional
assessment of the phage cream’s capacity to kill underlying bacteria in vitro, as the latter may be a
more realistic depiction of the capacity of the phages to be released from such cream formulations.

5. Lipophilicity of the Semi-Solid Base to Be Used for Phage Formulations

Hydrophobic ointments may potentially be useful in delivery of phage therapy to wounds and
ulcers, where their capacity to adhere to the wound and occlusive nature may be more appropriate
than formulations which are more hydrophilic. Quantitative assessment of the capacity for lipophilic
ointments to stably maintain viable phage numbers over time is not as straightforward as for creams.
This is because, unlike hydrophilic creams, the ointments do not solubilise and disperse evenly in
an aqueous solution, thus making the determination of plaque forming particles per unit volume
difficult [31]. Thus, experiments have focussed on qualitative assessment of phage lytic activity
following storage over time [31]. Whereas P. acnes phage creams demonstrated lytic capacity for at
least 90 days, the more hydrophobic emulsifying ointment and simple ointment white were capable of
supporting P. acnes phage lytic activity for shorter periods of time. For the simple ointment white, this
Pharmaceuticals 2018, 11, 26 8 of 12

was 50 days following storage at 4 ◦ C in light-protected containers, while storage at 45 ◦ C or constant

light exposure yielded no activity by 14 days. For the P. acnes phages formulated in emulsifying
ointment, phage efficacy was lost by seven days, even after storage at 4 ◦ C [31].
It is possible to speculate why these hydrophobic bases (emulsifying ointment in particular) do
not apparently support P. acnes phage lytic activity as do the more hydrophilic bases. It is possible that
the hydrophobic components in the emulsifying ointment may not partition well with aqueous phases
such as the aqueous PBS which is the carrier for the phages in these experiments. Another issue is that
the emulsifying ointment contains sodium lauryl sulphate, a surfactant capable of denaturing protein
and which has been shown to act as a virucide [49]. In this formulation, it may have contributed
to disruption of the phage protein coat. While sodium lauryl sulphate is also a component in the
formulation of aqueous cream, the concentration in emulsifying ointment is more than three times
higher [50]. This could explain why the phage viability is not as dramatically affected in aqueous
cream compared to emulsifying ointment (aqueous cream has previously been mentioned as capable of
supporting phage viability for at least 90 days) [31]. Another issue is that the thickness of the ointments
may have an inhibitory effect on phage release, as compared to the creams. In support of this possibility,
it has been noted that the composition of an agar plate surface can affect the morphology of phage
plaques, suggesting that a reduced capacity to form extensive clearing zones may reflect physical
barriers within the media [51].
Semi-solid formulations which are thicker than ointments, such as pastes, may not be optimal
bases for the delivery of phage for therapy. For instance, P. acnes phages formulated into zinc paste did
not show capacity to lyse surrounding bacteria in an in vitro model [31]. To investigate this further,
P. acnes phages were formulated alone into white soft paraffin (a component of zinc paste), as well
as white soft paraffin with the other components of zinc paste: starch and zinc oxide. Lysis of the
P. acnes bacteria was observed with the white soft paraffin alone and white soft paraffin containing
starch formulations [31]. White soft paraffin is a relatively unreactive substance [29], and despite its
hydrophobicity, did not adversely affect P. acnes phage release and lytic capacity. Starch appeared to
mildly inhibit release of the phage, possibly because of the thickness it imparts (as described above),
and the potential for starch macromolecules to entangle phage structures such as tails. The zinc oxide
was inhibitive of P. acnes phage release, possibly because of the sorption of phages to the surface of
zinc oxide particles. Metal oxide particles are known to form strong surface complexes with large
organic and charged macro-molecules, proteins and even bacteria [52].

6. Solid Formulations for the Delivery of Phages

While there are reports of availability of commercial solid dosage forms for delivery of phages
to infections of the epithelia [14,17,53], there is scant scientific literature on testing for efficacy and
phage stability of such products. Dosage forms such as troches offer potentially attractive approaches
to treating bacterial infections of the oral cavity, oesophagus, stomach and subsequent intestinal
tracts. Preparation of phages in aqueous solutions are potentially useful in the treatment of oral
infections [12,17,54,55]. However, delivery of phages over a sustained period would be optimal, and
troches as a vehicle offer such a delivery profile. Indeed, research into treatment of oral infections
suggests that application of medicaments in delivery forms such as troches is preferable to delivery
via suspensions or solutions [56], which are subject to inherent variability in exposure of the drug
to the oral cavity prior to patients swallowing or releasing the fluid from the mouth. While solid
substances at 4 ◦ C, troches begin to melt at temperatures approaching 37 ◦ C, and testing for the efficacy
and stability of phage troches has shown that at 37 ◦ C, the temperature which is found in the oral
cavity, these formulations are able to release lytic Klebsiella oxytoca (Myoviridae) and Rhodococcus equi
(Siphoviridae) phages in an in-vitro model, and that the phage lytic capacity was maintained for at least
49 days when troches were stored at 4 ◦ C [31,42].
It is important in these formulations that the phage particles are homogenously distributed
throughout the final product. This requires particular attention, as, unlike semi-solid preparations
Pharmaceuticals 2018, 11, 26 9 of 12

which can be triturated during and after addition of phages at room temperature, the troche formulation
is heated to approximately 60 ◦ C (a temperature that would inactivate most phages) in order to allow
miscibility of the constituents, and then solidifies upon cooling to temperatures of approximately
25 ◦ C (Professional Compounding Chemists of Australia). Therefore, while still molten, but following
cooling to temperatures which will minimise damage to the phage particles, the phages and the
formulation need to be mixed until homogenous, then poured into moulds and allowed to set.
The ingestion of troches with lytic phages as the medicament could prove to be a useful way
to treat bacterial infections within the stomach, or to modulate intestinal flora. Research has shown
that K. oxytoca Myoviridae phages are capable of surviving in environments simulating the gastric
chamber. Specifically, following 30 min exposure of 108 PFU/mL of K. oxytoca phages to simulated
gastric fluid at pH 2.5, in which time the phages may be expected to have passed through the stomach
if ingested via a troche, there were still significant numbers (105 –106 PFU/mL) of viable K. oxytoca
phages detected [42]. As low gastric pH significantly reduces phage numbers, the co-administration of
a proton pump inhibitor (PPI) drug, such as rabeprazole, which may have lower incidence of Phase
I/II drug interactions than other PPI, which have been used to assist in phage gastric survival [57],
may enhance phage viability. Such medications are very commonly used for gastric reflux, and have
the capacity to decrease stomach acidity to pH 6 [58]. Yet while in some patients PPI may increase the
risk of infections such as gastroenteritis [59] and pneumonia [60], this approach may alleviate the need
for microencapsulation of phages, which is being considered as a method of formulating phages for
improved viability in the gastric chamber, as well as to retain a high infective dose in the targeting of
a range of tissues downstream [61,62]. Once viable phages have passed through the stomach, they
will encounter bile salts in the duodenum, but as has been shown, they are not likely to be adversely
affected by such emulsifying agents [42,63,64].
For the delivery of phage therapy to infections in the lower intestinal and genito-urinary tracts,
suppositories/pessaries are useful vehicles [27]. As with the preparation of troches, formulation of
suppositories requires heating to high temperatures (100 ◦ C) to allow dissolution of constituents,
then cooling for pouring into moulds. Therefore, phages need to be added and thoroughly triturated
prior to solidification, but following cooling to temperatures which will not destroy the viral particles.
K. oxytoca (Myoviridae) and R. equi (Siphoviridae) phages formulated into these delivery forms have been
shown to be stable and viable in in-vitro models for at least 56 days when stored at 4 ◦ C [31,42].
In conclusion, a range of formulations are capable of successfully delivering lytic phage particles
for bacterial killing in vitro. These include semi-solid preparations such as creams and ointments, as
well as solid preparations such a troches and suppositories/pessaries. Phages are most stable when
stored at 4 ◦ C in these preparations, and remain active for at least 90 days in some formulations.
Factors which may influence the efficacy of phages in semi-solid preparations include the ionic nature
of the cream, as well as the thickness and stiffness of the formulations. Finally, the standardisation of
methodologies for assessment of efficacy of phages in these formulations is required.

Author Contributions: T.L.B., S.P., H.T.C., M.J.A. and J.T. wrote and reviewed the paper.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Spellberg, B.; Bartlett, J.G.; Gilbert, D.N. The future of antibiotics and resistance. N. Engl. J. Med. 2013, 368,
299–302. [CrossRef] [PubMed]
2. Aryee, A.; Price, N. Antimicrobial stewardship—Can we afford to do without it? Br. J. Clin. Pharmacol. 2015,
79, 173–181. [CrossRef] [PubMed]
3. Cosgrove, S.E.; Carmeli, Y. The impact of antimicrobial resistance on health and economic outcomes.
Clin. Infect. Dis. 2003, 36, 1433–1437. [PubMed]
4. Neu, H.C. The crisis in antibiotic resistance. Science 1992, 257, 1064–1073. [CrossRef] [PubMed]
Pharmaceuticals 2018, 11, 26 10 of 12

5. Levy, S.B.; Marshall, B. Antibacterial resistance worldwide: Causes, challenges and responses. Nat. Med.
2004, 10, 122–129. [CrossRef] [PubMed]
6. Schuts, E.C.; Hulscher, M.E.; Mouton, J.W.; Verduin, C.M.; Stuart, J.W.; Overdiek, H.W.; van der Linden, P.D.;
Natsch, S.; Hertogh, C.M.; Wolfs, T.F.; et al. Current evidence on hospital antimicrobial stewardship
objectives: A systematic review and meta-analysis. Lancet Infect. Dis. 2016, 16, 847–856. [CrossRef]
7. Sulakvelidze, A.; Alavidze, Z.; Morris, J.G., Jr. Bacteriophage therapy. Antimicrob. Agents Chemother. 2001, 45,
649–659. [CrossRef] [PubMed]
8. Cisek, A.A.; Dabrowska, I.; Gregorczyk, K.P.; Wyzewski, Z. Phage therapy in bacterial infections treatment:
One hundred years after the discovery of bacteriophages. Curr. Microbiol. 2017, 74, 277–283. [CrossRef]
[PubMed]
9. Bruynoghe, R.; Maisin, J. Essais de therapeutique au moyen du bacteriophage du staphylocoque. J. Compt.
Rend Soc. Biol. 1921, 85, 1020–1121.
10. Chanishvili, N. Phage therapy-history from twort and d'herelle through soviet experience to current
approaches. Adv. Virus Res. 2012, 83, 3–40. [PubMed]
11. Debarbieux, L.; Pirnay, J.P.; Verbeken, G.; De Vos, D.; Merabishvili, M.; Huys, I.; Patey, O.; Schoonjans, D.;
Vaneechoutte, M.; Zizi, M.; et al. A bacteriophage journey at the european medicines agency. Microbiol. Lett.
2016, 363. [CrossRef] [PubMed]
12. Vandenheuvel, D.; Lavigne, R.; Brussow, H. Bacteriophage therapy: Advances in formulation strategies and
human clinical trials. Annu. Rev. Virol. 2015, 2, 599–618. [CrossRef] [PubMed]
13. Loc-Carrillo, C.; Abedon, S.T. Pros and cons of phage therapy. Bacteriophage 2011, 1, 111–114. [CrossRef]
[PubMed]
14. Kutter, E.; De Vos, D.; Gvasalia, G.; Alavidze, Z.; Gogokhia, L.; Kuhl, S.; Abedon, S.T. Phage therapy in
clinical practice: Treatment of human infections. Curr. Pharm. Biotechnol. 2010, 11, 69–86. [CrossRef]
[PubMed]
15. NIH. New NIH Awards Will Support Development of Therapeutic Alternatives to Traditional Antibiotics.
Available online: https://www.niaid.nih.gov/news-events/new-nih-awards-will-support-development-
therapeutic-alternatives-traditional-antibiotics (accessed on 16 November 2017).
16. Chaplin, S. Topical antibacterial and antiviral agents: Prescribing and resistance. Prescriber 2016, 27, 29–36.
[CrossRef]
17. Abedon, S.T.; Kuhl, S.J.; Blasdel, B.G.; Kutter, E.M. Phage treatment of human infections. Bacteriophage 2011,
1, 66–85. [CrossRef] [PubMed]
18. Gu, J.; Liu, X.; Li, Y.; Han, W.; Lei, L.; Yang, Y.; Zhao, H.; Gao, Y.; Song, J.; Lu, R.; et al. A method for
generation phage cocktail with great therapeutic potential. PLoS ONE 2012, 7, e31698. [CrossRef] [PubMed]
19. Tothova, L.; Celec, P.; Babickova, J.; Gajdosova, J.; Al-Alami, H.; Kamodyova, N.; Drahovska, H.;
Liptakova, A.; Turna, J.; Hodosy, J. Phage therapy of cronobacter-induced urinary tract infection in mice.
Med. Sci. Monit. 2011, 17, BR173–BR178. [CrossRef] [PubMed]
20. Dufour, N.; Clermont, O.; La Combe, B.; Messika, J.; Dion, S.; Khanna, V.; Denamur, E.; Ricard, J.D.;
Debarbieux, L. Bacteriophage LM33_P1, a fast-acting weapon against the pandemic ST131-O25b:H4
Escherichia coli clonal complex. J. Antimicrob. Chemother. 2016, 71, 3072–3080. [CrossRef] [PubMed]
21. Laanto, E.; Bamford, J.K.; Ravantti, J.J.; Sundberg, L.R. The use of phage FCL-2 as an alternative to
chemotherapy against columnaris disease in aquaculture. Front. Microbiol. 2015, 6, 829. [CrossRef] [PubMed]
22. Kishor, C.; Mishra, R.R.; Saraf, S.K.; Kumar, M.; Srivastav, A.K.; Nath, G. Phage therapy of staphylococcal
chronic osteomyelitis in experimental animal model. Indian J. Med. Res. 2016, 143, 87–94. [PubMed]
23. Gu, J.; Li, X.; Yang, M.; Du, C.; Cui, Z.; Gong, P.; Xia, F.; Song, J.; Zhang, L.; Li, J.; et al. Therapeutic effect of
pseudomonas aeruginosa phage YH30 on mink hemorrhagic pneumonia. Vet. Microbiol. 2016, 190, 5–11.
[CrossRef] [PubMed]
24. Keen, E.C. Phage therapy: Concept to cure. Front. Microbiol. 2012, 3, 238. [CrossRef] [PubMed]
25. Cooper, C.J.; Khan Mirzaei, M.; Nilsson, A.S. Adapting drug approval pathways for bacteriophage-based
therapeutics. Front. Microbiol. 2016, 7, 1209. [CrossRef] [PubMed]
26. Brayfield, A. Martindale: The Complete Drug Reference; Pharmaceutical Press: London, UK, 2014.
27. Allen, L.V. Remington: An. Introduction to Pharmacy; Pharmaceutical Press: London, UK, 2013.
28. Aulton, M.E. Aulton's Pharmaceutics: The Design and Manufacture of Medicine; Churchill Livingstone:
Edinburgh, IN, USA, 2007.
Pharmaceuticals 2018, 11, 26 11 of 12

29. British Pharmacopoeia Commission. British Pharmacopoeia; Stationery Office: London, UK, 2012.
30. Alyami, H.; Dahmash, E.; Bowen, J.; Mohammed, A.R. An investigation into the effects of excipient particle
size, blending techniques and processing parameters on the homogeneity and content uniformity of a blend
containing low-dose model drug. PLoS ONE 2017, 12, e0178772. [CrossRef] [PubMed]
31. Brown, T.L.; Thomas, T.; Odgers, J.; Petrovski, S.; Spark, M.J.; Tucci, J. Bacteriophage formulated into a range
of semisolid and solid dosage forms maintain lytic capacity against isolated cutaneous and opportunistic
oral bacteria. J. Pharm. Pharmacol. 2017, 69, 244–253. [CrossRef] [PubMed]
32. O'Flaherty, S.; Ross, R.; Meaney, W.; Fitzgerald, G.; Elbreki, M.; Coffey, A. Potential of the polyvalent
anti-staphylococcus bacteriophage K for control of antibiotic-resistant staphylococci from hospitals.
Appl. Environ. Microbiol. 2005, 71, 1836–1842. [CrossRef] [PubMed]
33. Chen, L.-K.; Liu, Y.-L.; Hu, A.; Chang, K.-C.; Lin, N.-T.; Lai, M.-J.; Tseng, C.-C. Potential of bacteriophage
ΦAB2 as an environmental biocontrol agent for the control of multidrug-resistant Acinetobacter baumannii.
BMC Microbiol. 2013, 13, 154. [CrossRef] [PubMed]
34. Merabishvili, M.; Monserez, R.; van Belleghem, J.; Rose, T.; Jennes, S.; De Vos, D.; Verbeken, G.;
Vaneechoutte, M.; Pirnay, J.-P. Stability of bacteriophages in burn wound care products. PLoS ONE 2017,
12, e0182121. [CrossRef] [PubMed]
35. Sansom, C. Phage therapy for severe infections tested in the first multicentre trial. Lancet Infect. Dis. 2015, 15,
1384–1385. [CrossRef]
36. NIH. National Institutes of Health Clinical Trials. Available online: https://clinicaltrials.gov/ct2/show/
NCT02116010?term=NCT02116010&rank=1 (accessed on 14 June 2017).
37. Brown, T.L.; Petrovski, S.; Dyson, Z.A.; Seviour, R.; Tucci, J. The formulation of bacteriophage in a semi
solid preparation for control of Propionibacterium acnes growth. PLoS ONE 2016, 11, e0151184. [CrossRef]
[PubMed]
38. Biemer, J.J. Antimicrobial susceptibility testing by the kirby-bauer disc diffusion method. Ann. Clin. Lab. Sci.
1973, 3, 135–140. [PubMed]
39. Hudzicki, J. Kirby-Bauer Disk Diffusion Suseptibility Test Protocol. Available online: http://www.
asmscience.org/content/education/protocol/protocol.3189 (Accessed on 18 December 2017).
40. European Committee on Antimicrobial Suseptability Testing. Eucast Disk Diffusion Test Methodology.
Available online: http://www.eucast.org/ast_of_bacteria/disk_diffusion_methodology/ (accessed on 18
December 2017).
41. U.S. Department of Heath and Human Services Food and Drug Administration. Guide for Industry:
Potency Test for Cellular and Gene Therapy Products. Available online: https://www.fda.gov/
downloads/biologicsbloodvaccines/guidancecomplianceregulatoryinformation/guidances/
cellularandgenetherapy/ucm243392.pdf (accessed on 18 December 2017).
42. Brown, T.L.; Petrovski, S.; Hoyle, D.; Chan, H.T.; Lock, P.; Tucci, J. Characterization and formulation into
solid dosage forms of a novel bacteriophage lytic against klebsiella oxytoca. PLoS ONE 2017, 12, e0183510.
[CrossRef] [PubMed]
43. Brown, T.L.; Tucci, J.; Dyson, Z.A.; Lock, P.; Adda, C.G.; Petrovski, S. Dynamic interactions between
prophages induce lysis in propionibacterium acnes. Res. Microbiol. 2017, 168, 103–112. [CrossRef] [PubMed]
44. Todd, C. On the electrical behaviour of the bacteriophage. Brit. J. Exp. Path. 1927, 8, 369–376.
45. Serwer, P.; Pichler, M.E. Electrophoresis of bacteriophage T7 and T7 capsids in agarose gels. J. Virol. 1978, 28,
917–928. [PubMed]
46. Serwer, P.; Hayes, S.J. Agarose gel electrophoresis of bacteriophages and related particles. I. Avoidance of
binding to the gel and recognizing of particles with packaged DNA. Electrophoresis 1982, 3, 76–80. [CrossRef]
47. Cademartiri, R.; Anany, H.; Gross, I.; Bhayani, R.; Griffiths, M.; Brook, M.A. Immobilization of bacteriophages
on modified silica particles. Biomaterials 2010, 31, 1904–1910. [CrossRef] [PubMed]
48. Anany, H.; Chen, W.; Pelton, R.; Griffiths, M.W. Biocontrol of listeria monocytogenes and Escherichia coli
O157:H7 in meat by using phages immobilized on modified cellulose membranes. Appl. Environ. Microbiol.
2011, 77, 6379–6387. [CrossRef] [PubMed]
49. Piret, J.; Desormeaux, A.; Bergeron, M.G. Sodium lauryl sulfate, a microbicide effective against enveloped
and nonenveloped viruses. Curr. Drug Targets 2002, 3, 17–30. [CrossRef] [PubMed]
50. Sansom, L. Australian Pharmaceutical Formulary and Handbook, 23rd ed.; Pharmaceutical Society of Australia:
Canberra, Australia, 2015; pp. 36–48.
Pharmaceuticals 2018, 11, 26 12 of 12

51. Abedon, S.T.; Yin, J. Bacteriophage plaques: Theory and analysis. Methods Mol. Biol. 2009, 501, 161–174.
[PubMed]
52. Thai, C.K.; Dai, H.; Sastry, M.S.; Sarikaya, M.; Schwartz, D.T.; Baneyx, F. Identification and characterization
of cu(2)o- and zno-binding polypeptides by escherichia coli cell surface display: Toward an understanding
of metal oxide binding. Biotechnol. Bioeng. 2004, 87, 129–137. [CrossRef] [PubMed]
53. Kutateladze, M.; Adamia, R. Phage therapy experience at the eliava institute. Med. Mal. Infect. 2008, 38,
426–430. [CrossRef] [PubMed]
54. Ryan, E.M.; Gorman, S.P.; Donnelly, R.F.; Gilmore, B.F. Recent advances in bacteriophage therapy:
How delivery routes, formulation, concentration and timing influence the success of phage therapy.
J. Pharm. Pharmacol. 2011, 63, 1253–1264. [CrossRef] [PubMed]
55. Fish, R.; Kutter, E.; Wheat, G.; Blasdel, B.; Kutateladze, M.; Kuhl, S. Bacteriophage treatment of intransigent
diabetic toe ulcers: A case series. J. Wound Care 2016, 25, S27–S33. [CrossRef] [PubMed]
56. Lyu, X.; Zhao, C.; Yan, Z.M.; Hua, H. Efficacy of nystatin for the treatment of oral candidiasis: A systematic
review and meta-analysis. Drug Des. Devel. Ther. 2016, 10, 1161–1171. [CrossRef] [PubMed]
57. Miedzybrodzki, R.; Klak, M.; Jonczyk-Matysiak, E.; Bubak, B.; Wojcik, A.; Kaszowska, M.; Weber-Dabrowska, B.;
Lobocka, M.; Gorski, A. Means to facilitate the overcoming of gastric juice barrier by a therapeutic staphylococcal
bacteriophage a5/80. Front. Microbiol. 2017, 8, 467. [CrossRef] [PubMed]
58. Netzer, P.; Gaia, C.; Sandoz, M.; Huluk, T.; Gut, A.; Halter, F.; Husler, J.; Inauen, W. Effect of repeated injection
and continuous infusion of omeprazole and ranitidine on intragastric pH over 72 h. Am. J. Gastroenterol.
1999, 94, 351–357. [PubMed]
59. Tennant, S.M.; Hartland, E.L.; Phumoonna, T.; Lyras, D.; Rood, J.I.; Robins-Browne, R.M.; van Driel, I.R.
Influence of gastric acid on susceptibility to infection with ingested bacterial pathogens. Infect. Immun. 2008,
76, 639–645. [CrossRef] [PubMed]
60. Laheij, R.J.; Sturkenboom, M.C.; Hassing, R.J.; Dieleman, J.; Stricker, B.H.; Jansen, J.B. Risk of
community-acquired pneumonia and use of gastric acid-suppressive drugs. JAMA 2004, 292, 1955–1960.
[CrossRef] [PubMed]
61. Malik, D.J.; Sokolov, I.J.; Vinner, G.K.; Mancuso, F.; Cinquerrui, S.; Vladisavljevic, G.T.; Clokie, M.R.J.;
Garton, N.J.; Stapley, A.G.F.; Kirpichnikova, A. Formulation, stabilisation and encapsulation of bacteriophage
for phage therapy. Adv. Colloid Interface Sci. 2017, 249, 100–133. [CrossRef] [PubMed]
62. Ma, Y.; Pacan, J.C.; Wang, Q.; Xu, Y.; Huang, X.; Korenevsky, A.; Sabour, P.M. Microencapsulation of
bacteriophage felix O1 into chitosan-alginate microspheres for oral delivery. Appl. Environ. Microbiol. 2008,
74, 4799–4805. [CrossRef] [PubMed]
63. Koo, J.; DePaola, A.; Marshall, D.L. Effect of simulated gastric fluid and bile on survival of vibrio vulnificus
and vibrio vulnificus phage. J. Food Prot. 2000, 63, 1665–1669. [CrossRef] [PubMed]
64. Verthe, K.; Possemiers, S.; Boon, N.; Vaneechoutte, M.; Verstraete, W. Stability and activity of an enterobacter
aerogenes-specific bacteriophage under simulated gastro-intestinal conditions. Appl. Microbiol. Biotechnol.
2004, 65, 465–472. [CrossRef] [PubMed]

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